In the past, the inventors herein have patented a device for the staining of biological samples in microgravity aboard a spacecraft, known as the centrifuge operated slide stainer. This device and technology was developed in response to the need for real-time analysis of crew member blood samples and microbiological monitoring of samples obtained from the environmental systems of a spacecraft. That method and associated apparatus, hereinafter COSS, appear in U.S. Pat. No. 6,008,009, dated Dec. 28, 1999, entitled Centrifuge-Operated Specimen Staining Method and Apparatus, inventors Mark S. F. Clarke and Daniel L. Feeback. Further study of the need for analysis of biological samples during space flight suggested that a greater range of sample types and staining protocols would have to be accommodated by any staining technology selected for flight. This was due not only for medical operations reasons and environmental monitoring, but also for the anticipated increased requirement for analysis of biological samples obtained from science experiments conducted aboard the established International Space Station. In order to accommodate such an increase in demand, the possibility of miniaturizing the COSS technology was investigated so that a greater range of samples and staining protocols could be accommodated with no, or less, impact on crew time, solid waste production and upmass than that of the aforesaid technology. The need for real-time analysis of biological samples during space flight is exemplified by the requirement for a differential white cell count DWCC, a critical medical diagnostic tool which can be used to distinguish between various conditions that induce alterations in the total number and type of white blood cells produced by the human body. For example, a DWCC can be used to distinguish between bacterial or viral infections, in the differential diagnosis of an allergic reaction or to detect the presence of myeloproliferative disorders or leukemia. Microgravity exposure during space flight results in hemodynamic changes in crew members, which in turn impacts the production of white blood cells. Heretofore, no data are available to establish the xe2x80x9cnormal baselinexe2x80x9d for white blood cell production in microgravity. Without first knowing the extent to which microgravity exposure impacts white blood cell production, or secondly the proper xe2x80x9cmicrogravity baselinexe2x80x9d for a normal healthy crew member in space, it is quite possible that a bacterial or viral infection may be overlooked or misdiagnosed, or that a potentially much more serious problem, such as leukemia, may be attributed to a bacterial or viral infection in a particular crew member. In addition, the requirement for microbiological screening of both medical and environmental samples during space flight, which can only be accomplished by utilizing specific staining techniques for microbe identification, is a second example of the need for real-time analysis capabilities using standard staining techniques on-orbit. At present, it is impossible to perform a DWCC while aboard a space craft. Whole blood smears have been produced in microgravity, but as yet it has remained impossible to perform a DWCC without returning the blood smear to Earth. Due to the limited life span of such smears, it is impossible to make a definitive statement with regard to the effect of microgravity exposure upon white blood cell profiles based on such samples. Until real-time performance and analysis of a DWCC can be achieved aboard the space craft, critical crew health information thus remains unobtainable.
In a terrestrial setting, a differential white cell count is obtained by preparing a blood smear on a glass slide, fixing the cells in the smear to the surface of the slide, staining the cells with a histochemical stain followed by washing the slide in a clean buffer solution prior to viewing under the microscope where a xe2x80x9cdifferentialxe2x80x9d white blood cell count is made by morphological criteria. The protocol outlined above is a simple and universally used technique to perform a DWCC. However, this technique requires the use of liquid buffer solutions, including fixatives and dye solutions. While this technique is performed easily on Earth, the problems associated with liquid handling in microgravity make such a task nearly impossible. Past attempts at solving this problem have included several xe2x80x9ccell stainersxe2x80x9d which were tested by NASA or its contractor personnel but have since proved unsuitable for use in microgravity. The first attempt was a slide stainer which flew aboard Sky Lab. This device proved very cumbersome, required large volumes of buffer solutions and had limited use due to precipitate formation in the staining solutions which blocked the intricate tubing arrangement required to apply the staining solutions to the blood smear. A second attempt was based upon an airtight chamber design which contained a blood smear slide, into which buffer solutions and/or staining solutions were introduced using a vacuum system. System operation relied upon a series of one-way and two-way valves in order to achieve an efficient vacuum into which the staining solutions were introduced by hypodermic syringe. The original technology used a hand-held squeeze bulb to create the vacuum which proved inadequate. A later version incorporated mechanical pumps to provide both vacuum production and syringe emptying. The hand-operated version of this technology, although shown to work on the ground and which passed initial testing aboard the KC-135 parabolic aircraft, did not fulfill its potential and has since been shelved as a viable solution to slide staining on-orbit, not least because of its requirement for substantial crew interaction and crew time.
In the present technology, termed Directional Acceleration Vector-Driven Displacement of Fluids DAVD-DOF the same end point, namely sequential filling and emptying of a staining chamber, is achieved using a network of reservoirs and connecting tubes created on a single slide. However, unlike the earlier COSS technology, fluid displacement is achieved by utilizing the weight of the fluid itself, rather than a weighted plunger, to force the fluid through a network of channels between fluid reservoirs. Selective emptying of separate fluid reservoirs is achieved by altering the cross-sectional area of the channel which connects the reservoirs. As cross-sectional area of the channel decreases, the g-force required to bring about fluid displacement through the channel is increased. This approach reduces the overall size of the equipment required to perform a staining protocol in microgravity as well as reducing the amount of staining reagent required from approximately 3 milliliters per reagent in the original COSS technology to less than 20 microliters in the DAVD-DOF technology. As the staining protocol is carried out in a centrifuge at g-levels above 1xc3x97 g, the problems associated with liquid handling in microgravity, such as air/liquid mixing and bubble formation do not occur. This is due to the fact that a liquid is much heavier than air in the increased acceleration field produced by its rotation in a centrifuge, thereby producing a clear and defined liquid/air interface, an attribute common to both the original COSS and present DAVD-DOF technologies. The technology described herein is thus based upon the concept that fluids, in this case, staining reagents used for biological sample analysis, can be transferred from one reservoir to another through a connecting tube/channel by applying a gravity vector or acceleration vector in the direction of the required movement. This concept is essentially different from the original COSS device, U.S. Pat. No. 6,008,009. That system utilizes a weighted plunger designed to force fluid from one container to another at a constant level of hypergravity maintained in a standard swing-bucket centrifuge. This arrangement allows the sequential filling and emptying of a staining chamber containing a microscope slide on which a biological sample is mounted. In this centrifugal analysis, the principle of invention is stated as optimizing defined dimensional channels of a specimen slide to effect controlled movement of specimen fluids therein by defined g-forces.